Broad proteomics analysis of seeding-induced aggregation of α-synuclein in M83 neurons reveals remodeling of proteostasis mechanisms that might contribute to Parkinson's disease pathogenesis.

Aggregation of misfolded α-synuclein (α-syn) is a key characteristic feature of Parkinson's disease (PD) and related synucleinopathies. The nature of these aggregates and their contribution to cellular dysfunction is still not clearly elucidated. We employed mass spectrometry-based total and phospho-proteomics to characterize the underlying molecular and biological changes due to α-syn aggregation using the M83 mouse primary neuronal model of PD. We identified gross changes in the proteome that coincided with the formation of large Lewy body-like α-syn aggregates in these neurons. We used protein-protein interaction (PPI)-based network analysis to identify key protein clusters modulating specific biological pathways that may be dysregulated and identified several mechanisms that regulate protein homeostasis (proteostasis). The observed changes in the proteome may include both homeostatic compensation and dysregulation due to α-syn aggregation and a greater understanding of both processes and their role in α-syn-related proteostasis may lead to improved therapeutic options for patients with PD and related disorders.

Meta-Analysis of the Alzheimer's Disease Human Brain Transcriptome and Functional Dissection in Mouse Models.

We present a consensus atlas of the human brain transcriptome in Alzheimer's disease (AD), based on meta-analysis of differential gene expression in 2,114 postmortem samples. We discover 30 brain coexpression modules from seven regions as the major source of AD transcriptional perturbations. We next examine overlap with 251 brain differentially expressed gene sets from mouse models of AD and other neurodegenerative disorders. Human-mouse overlaps highlight responses to amyloid versus tau pathology and reveal age- and sex-dependent expression signatures for disease progression. Human coexpression modules enriched for neuronal and/or microglial genes broadly overlap with mouse models of AD, Huntington's disease, amyotrophic lateral sclerosis, and aging. Other human coexpression modules, including those implicated in proteostasis, are not activated in AD models but rather following other, unexpected genetic manipulations. Our results comprise a cross-species resource, highlighting transcriptional networks altered by human brain pathophysiology and identifying correspondences with mouse models for AD preclinical studies.

Harnessing peripheral DNA methylation differences in the Alzheimer's Disease Neuroimaging Initiative (ADNI) to reveal novel biomarkers of disease.

BACKGROUND: Alzheimer's disease (AD) is a chronic progressive neurodegenerative disease impacting an estimated 44 million adults worldwide. The causal pathology of AD (accumulation of amyloid-beta and tau), precedes hallmark symptoms of dementia by more than a decade, necessitating development of early diagnostic markers of disease onset, particularly for new drugs that aim to modify disease processes. To evaluate differentially methylated positions (DMPs) as novel blood-based biomarkers of AD, we used a subset of 653 individuals with peripheral blood (PB) samples in the Alzheimer's disease Neuroimaging Initiative (ADNI) consortium. The selected cohort of AD, mild cognitive impairment (MCI), and age-matched healthy controls (CN) all had imaging, genetics, transcriptomics, cerebrospinal protein markers, and comprehensive clinical records, providing a rich resource of concurrent multi-omics and phenotypic information on a well-phenotyped subset of ADNI participants. RESULTS: In this manuscript, we report cross-diagnosis differential peripheral DNA methylation in a cohort of AD, MCI, and age-matched CN individuals with longitudinal DNA methylation measurements. Epigenome-wide association studies (EWAS) were performed using a mixed model with repeated measures over time with a P value cutoff of 1 × 10-5 to test contrasts of pairwise differential peripheral methylation in AD vs CN, AD vs MCI, and MCI vs CN. The most highly significant differentially methylated loci also tracked with Mini Mental State Examination (MMSE) scores. Differentially methylated loci were enriched near brain and neurodegeneration-related genes (e.g., BDNF, BIN1, APOC1) validated using the genotype tissue expression project portal (GTex). CONCLUSIONS: Our work shows that peripheral differential methylation between age-matched subjects with AD relative to healthy controls will provide opportunities to further investigate and validate differential methylation as a surrogate of disease. Given the inaccessibility of brain tissue, the PB-associated methylation marks may help identify the stage of disease and progression phenotype, information that would be central to bringing forward successful drugs for AD.

The Major Risk Factors for Alzheimer's Disease: Age, Sex, and Genes Modulate the Microglia Response to Aß Plaques.

Gene expression profiles of more than 10,000 individual microglial cells isolated from cortex and hippocampus of male and female AppNL-G-F mice over time demonstrate that progressive amyloid-ß accumulation accelerates two main activated microglia states that are also present during normal aging. Activated response microglia (ARMs) are composed of specialized subgroups overexpressing MHC type II and putative tissue repair genes (Dkk2, Gpnmb, and Spp1) and are strongly enriched with Alzheimer's disease (AD) risk genes. Microglia from female mice progress faster in this activation trajectory. Similar activated states are also found in a second AD model and in human brain. Apoe, the major genetic risk factor for AD, regulates the ARMs but not the interferon response microglia (IRMs). Thus, the ARMs response is the converging point for aging, sex, and genetic AD risk factors.

Novel methods for integration and visualization of genomics and genetics data in Alzheimer's disease.

INTRODUCTION: Numerous omics studies have been conducted to understand the molecular networks involved in Alzheimer's disease (AD), but the pathophysiology is still not completely understood; new approaches that enable neuroscientists to better interpret the results of omics analysis are required. METHODS: We have developed advanced methods to analyze and visualize publicly-available genomics and genetics data. The tools include a composite clinical-neuropathological score for defining AD, gene expression maps in the brain, and networks integrating omics data to understand the impact of polymorphisms on AD pathways. RESULTS: We have analyzed over 50 public human gene expression data sets, spanning 19 different brain regions and encompassing three separate cohorts. We integrated genome-wide association studies with expression data to identify important genes in the pathophysiology of AD, which provides further insight into the calcium signaling and calcineurin pathways. DISCUSSION: Biologists can use these freely-available tools to obtain a comprehensive, information-rich view of the pathways in AD.

Reproductive period and epigenetic modifications of the oxidative phosphorylation pathway in the human prefrontal cortex.

PURPOSE: Human females have a unique duration of post-reproductive longevity, during which sex-specific mechanisms ma influence later-life mechanisms of neuronal resilience and vulnerability. The maintenance of energy metabolism, through the oxidative phosphorylation (OXPHOS) apparatus, is essential for brain health. Given the known association between reproductive period (years from menarche to menopause) and cognitive aging, we examined the hypothesis that cumulative estrogen exposure across the lifetime may be associated with differential methylation of genes in the OXPHOS pathway. METHODS: Using DNA methylation patterns in the post-mortem dorsolateral prefrontal cortex (DLPFC) of 426 women prospectively followed until death in the Religious Orders Study and Rush Memory and Aging Project, we examined the relationship between reproductive period (subtracting age at menarche from age at menopause) and DNA methylation of a published set of autosomal OXPHOS genes previously implicated in stroke susceptibility. We then performed an unsupervised analysis of methylation levels across the Hallmark pathways from the Molecular Signatures Database. RESULTS: We observed a strong association between reproductive period and DNA methylation status across OXPHOS CpGs. We replicated this association between reproductive period and DNA methylation in a much larger set of OXPHOS genes in our unsupervised analysis. Here, reproductive period also showed associations with methylation in genes related to E2F, MYC and MTORC1 signaling, fatty acid metabolism and DNA repair. CONCLUSION: This study provides evidence from both a supervised and unsupervised analyses, that lifetime cumulative endogenous steroid exposures may play a role in maintenance of post-menopausal cellular balance, including in brain tissue.

Water-deficit-induced changes in transcription factor expression in maize seedlings.

Plants tolerate water deficits by regulating gene networks controlling cellular and physiological traits to modify growth and development. Transcription factor (TF)-directed regulation of transcription within these gene networks is key to eliciting appropriate responses. In this study, reverse transcription quantitative PCR (RT-qPCR) was used to examine the abundance of 618 transcripts from 536 TF genes in individual root and shoot tissues of maize seedlings grown in vermiculite under well-watered (water potential of -0.02 MPa) and water-deficit conditions (water potentials of -0.3 and -1.6 MPa). A linear mixed model identified 433 TF transcripts representing 392 genes that differed significantly in abundance in at least one treatment, including TFs that intersect growth and development and environmental stress responses. TFs were extensively differentially regulated across stressed maize seedling tissues. Hierarchical clustering revealed TFs with stress-induced increased abundance in primary root tips that likely regulate root growth responses to water deficits, possibly as part of abscisic acid and/or auxin-dependent signaling pathways. Ten of these TFs were selected for validation in nodal root tips of drought-stressed field-grown plants (late V1 to early V2 stage). Changes in abundance of these TF transcripts under a field drought were similar to those observed in the seedling system.

The Role of MAPT Haplotype H2 and Isoform 1N/4R in Parkinsonism of Older Adults.

BACKGROUND AND OBJECTIVE: Recently, we have shown that the Parkinson's disease (PD) susceptibility locus MAPT (microtubule associated protein tau) is associated with parkinsonism in older adults without a clinical diagnosis of PD. In this study, we investigated the relationship between parkinsonian signs and MAPT transcripts by assessing the effect of MAPT haplotypes on alternative splicing and expression levels of the most common isoforms in two prospective clinicopathologic studies of aging. MATERIALS AND METHODS: using regression analysis, controlling for age, sex, study and neuropathology, we evaluated 976 subjects with clinical, genotyping and brain pathology data for haplotype analysis. For transcript analysis, we obtained MAPT gene and isoform-level expression from the dorsolateral prefrontal cortex for 505 of these subjects. RESULTS: The MAPT H2 haplotype was associated with lower total MAPT expression (p = 1.2x10-14) and global parkinsonism at both study entry (p = 0.001) and proximate to death (p = 0.050). Specifically, haplotype H2 was primarily associated with bradykinesia in both assessments (p<0.001 and p = 0.008). MAPT total expression was associated with age and decreases linearly with advancing age (p<0.001). Analysing MAPT alternative splicing, the expression of 1N/4R isoform was inversely associated with global parkinsonism (p = 0.008) and bradykinesia (p = 0.008). Diminished 1N/4R isoform expression was also associated with H2 (p = 0.001). CONCLUSIONS: Overall, our results suggest that age and H2 are associated with higher parkinsonism score and decreased total MAPT RNA expression. Additionally, we found that H2 and parkinsonism are associated with altered expression levels of specific isoforms. These findings may contribute to the understanding of the association between MAPT locus and parkinsonism in elderly subjects and in some extent to age-related neurodegenerative diseases.

Rheumatoid arthritis-associated RBPJ polymorphism alters memory CD4+ T cells.

Notch signaling has recently emerged as an important regulator of immune responses in autoimmune diseases. The recombination signal-binding protein for immunoglobulin kappa J region (RBPJ) is a transcriptional repressor, but converts into a transcriptional activator upon activation of the canonical Notch pathway. Genome-wide association studies of rheumatoid arthritis (RA) identified a susceptibility locus, rs874040(CC), which implicated the RBPJ gene. Here, chromatin state mapping generated using the chromHMM algorithm reveals strong enhancer regions containing DNase I hypersensitive sites overlapping the rs874040 linkage disequilibrium block in human memory, but not in naïve CD4(+) T cells. The rs874040 overlapping this chromatin state was associated with increased RBPJ expression in stimulated memory CD4(+) T cells from healthy subjects homozygous for the risk allele (CC) compared with memory CD4(+) T cells bearing the protective allele (GG). Transcriptomic analysis of rs874040(CC) memory T cells showed a repression of canonical Notch target genes IL (interleukin)-9, IL-17 and interferon (IFN)γ in the basal state. Interestingly, activation of the Notch pathway using soluble Notch ligand, Jagged2-Fc, induced IL-9 and IL-17A while delta-like 4Fc, another Notch ligand, induced higher IFNγ expression in the rs874040(CC) memory CD4(+) T cells compared with their rs874040(GG) counterparts. In RA, RBPJ expression is elevated in memory T cells from RA patients compared with control subjects, and this was associated with induced inflammatory cytokines IL-9, IL-17A and IFNγ in response to Notch ligation in vitro. These findings demonstrate that the rs874040(CC) allele skews memory T cells toward a pro-inflammatory phenotype involving Notch signaling, thus increasing the susceptibility to develop RA.

Alzheimer's loci: epigenetic associations and interaction with genetic factors.

OBJECTIVE: We explore the role of DNA methylation in Alzheimer's disease (AD). To elucidate where DNA methylation falls along the causal pathway linking risk factors to disease, we examine causal models to assess its role in the pathology of AD. METHODS: DNA methylation profiles were generated in 740 brain samples using the Illumina HumanMet450K beadset. We focused our analysis on CpG sites from 11 AD susceptibility gene regions. The primary outcome was a quantitative measure of neuritic amyloid plaque (NP), a key early element of AD pathology. We tested four causal models: (1) independent associations, (2) CpG mediating the association of a variant, (3) reverse causality, and (4) genetic variant by CpG interaction. RESULTS: Six genes regions (17 CpGs) showed evidence of CpG associations with NP, independent of genetic variation - BIN1 (5), CLU (5), MS4A6A (3), ABCA7 (2), CD2AP (1), and APOE (1). Together they explained 16.8% of the variability in NP. An interaction effect was seen in the CR1 region for two CpGs, cg10021878 (P = 0.01) and cg05922028 (P = 0.001), in relation to NP. In both cases, subjects with the risk allele rs6656401(AT) (/) (AA) display more methylation being associated with more NP burden, whereas subjects with the rs6656401(TT) protective genotype have an inverse association with more methylation being associated with less NP. INTERPRETATION: These observations suggest that, within known AD susceptibility loci, methylation is related to pathologic processes of AD and may play a largely independent role by influencing gene expression in AD susceptibility loci.

Association of DNA methylation in the brain with age in older persons is confounded by common neuropathologies.

DNA methylation plays a crucial role in the regulation of gene expression, cell differentiation and development. Previous studies have reported age-related alterations of methylation levels in the human brain across the lifespan, but little is known about whether the observed association with age is confounded by common neuropathologies among older persons. Using genome-wide DNA methylation data from 740 postmortem brains, we interrogated 420,132 CpG sites across the genome in a cohort of individuals with ages from 66 to 108 years old, a range of ages at which many neuropathologic indices become quite common. We compared the association of DNA methylation prior to and following adjustment for common neuropathologies using a series of linear regression models. In the simplest model adjusting for technical factors including batch effect and bisulfite conversion rate, we found 8156 CpGs associated with age. The number of CpGs associated with age dropped by more than 10% following adjustment for sex. Notably, after adjusting for common neuropathologies, the total number of CpGs associated with age was reduced by approximately 40%, compared to the sex-adjusted model. These data illustrate that the association of methylation changes in the brain with age is inflated if one does not account for age-related brain pathologies. This article is part of a Directed Issue entitled: Epigenetics dynamics in development and disease.

PRIMEGENSw3: a web-based tool for high-throughput primer and probe design.

Highly specific and efficient primer and probe design has been a major hurdle in many high-throughput techniques. Successful implementation of any PCR or probe hybridization technique depends on the quality of primers and probes used in terms of their specificity and cross-hybridization. Here we describe PRIMEGENSw3, a set of web-based utilities for high-throughput primer and probe design. These utilities allow users to select genomic regions and to design primer/probe for selected regions in an interactive, user-friendly, and automatic fashion. The system runs the PRIMEGENS algorithm in the back-end on the high-performance server with the stored genomic database or user-provided custom database for cross-hybridization check. Cross-hybridization is checked not only using BLAST but also by checking mismatch positions and energy calculation of potential hybridization hits. The results can be visualized online and also can be downloaded. The average success rate of primer design using PRIMEGENSw3 is ~90 %. The web server also supports primer design for methylated sequences, which is used in epigenetic studies. Stand-alone version of the software is also available for download at the website.

Association of Brain DNA methylation in SORL1, ABCA7, HLA-DRB5, SLC24A4, and BIN1 with pathological diagnosis of Alzheimer disease.

IMPORTANCE: Recent large-scale genome-wide association studies have discovered several genetic variants associated with Alzheimer disease (AD); however, the extent to which DNA methylation in these AD loci contributes to the disease susceptibility remains unknown. OBJECTIVE: To examine the association of brain DNA methylation in 28 reported AD loci with AD pathologies. DESIGN, SETTING, AND PARTICIPANTS: Ongoing community-based clinical pathological cohort studies of aging and dementia (the Religious Orders Study and the Rush Memory and Aging Project) among 740 autopsied participants 66.0 to 108.3 years old. EXPOSURES: DNA methylation levels at individual CpG sites generated from dorsolateral prefrontal cortex tissue using a bead assay. MAIN OUTCOMES AND MEASURES: Pathological diagnosis of AD by National Institute on Aging-Reagan criteria following a standard postmortem examination. RESULTS: Overall, 447 participants (60.4%) met the criteria for pathological diagnosis of AD. Brain DNA methylation in SORL1, ABCA7, HLA-DRB5, SLC24A4, and BIN1 was associated with pathological AD. The association was robustly retained after replacing the binary trait of pathological AD with 2 quantitative and molecular specific hallmarks of AD, namely, Aß load and paired helical filament tau tangle density. Furthermore, RNA expression of transcripts of SORL1 and ABCA7 was associated with paired helical filament tau tangle density, and the expression of BIN1 was associated with Aß load. CONCLUSIONS AND RELEVANCE: Brain DNA methylation in multiple AD loci is associated with AD pathologies. The results provide further evidence that disruption of DNA methylation is involved in the pathological process of AD.

Epigenomics of Alzheimer's disease.

Alzheimer's disease (AD) is a large and growing public health problem. It is characterized by the accumulation of amyloid ß peptides and abnormally phosphorylated tau proteins that are associated with cognitive decline and dementia. Much has been learned about the genomics of AD from linkage analyses and, more recently, genome-wide association studies. Several but not all aspects of the genomic landscape are involved in amyloid ß metabolism. The moderate concordance of disease among twins suggests other factors, potentially epigenomic factors, are related to AD. We are at the earliest stages of examining the relation of the epigenome to the clinical and pathologic phenotypes that characterize AD. Our literature review suggests that there is some evidence of age-related changes in human brain methylation. Unfortunately, studies of AD have been relatively small with limited coverage of methylation sites and microRNA, let alone other epigenomic marks. We are in the midst of 2 large studies of human brains including coverage of more than 420,000 autosomal cytosine-guanine dinucleotides with the Illumina Infinium HumanMethylation450 BeadArray, and histone acetylation with chromatin immunoprecipitation sequencing. We present descriptive data to help inform other researchers what to expect from these approaches to better design and power their studies. We then discuss future directions to inform on the epigenomic architecture of AD.

24-hour rhythms of DNA methylation and their relation with rhythms of RNA expression in the human dorsolateral prefrontal cortex.

Circadian rhythms modulate the biology of many human tissues, including brain tissues, and are driven by a near 24-hour transcriptional feedback loop. These rhythms are paralleled by 24-hour rhythms of large portions of the transcriptome. The role of dynamic DNA methylation in influencing these rhythms is uncertain. While recent work in Neurospora suggests that dynamic site-specific circadian rhythms of DNA methylation may play a role in modulating the fungal molecular clock, such rhythms and their relationship to RNA expression have not, to our knowledge, been elucidated in mammalian tissues, including human brain tissues. We hypothesized that 24-hour rhythms of DNA methylation exist in the human brain, and play a role in driving 24-hour rhythms of RNA expression. We analyzed DNA methylation levels in post-mortem human dorsolateral prefrontal cortex samples from 738 subjects. We assessed for 24-hour rhythmicity of 420,132 DNA methylation sites throughout the genome by considering methylation levels as a function of clock time of death and parameterizing these data using cosine functions. We determined global statistical significance by permutation. We then related rhythms of DNA methylation with rhythms of RNA expression determined by RNA sequencing. We found evidence of significant 24-hour rhythmicity of DNA methylation. Regions near transcription start sites were enriched for high-amplitude rhythmic DNA methylation sites, which were in turn time locked to 24-hour rhythms of RNA expression of nearby genes, with the nadir of methylation preceding peak transcript expression by 1-3 hours. Weak ante-mortem rest-activity rhythms were associated with lower amplitude DNA methylation rhythms as were older age and the presence of Alzheimer's disease. These findings support the hypothesis that 24-hour rhythms of DNA methylation, particularly near transcription start sites, may play a role in driving 24-hour rhythms of gene expression in the human dorsolateral prefrontal cortex, and may be affected by age and Alzheimer's disease.

Alzheimer's disease: early alterations in brain DNA methylation at ANK1, BIN1, RHBDF2 and other loci.

We used a collection of 708 prospectively collected autopsied brains to assess the methylation state of the brain's DNA in relation to Alzheimer's disease (AD). We found that the level of methylation at 71 of the 415,848 interrogated CpGs was significantly associated with the burden of AD pathology, including CpGs in the ABCA7 and BIN1 regions, which harbor known AD susceptibility variants. We validated 11 of the differentially methylated regions in an independent set of 117 subjects. Furthermore, we functionally validated these CpG associations and identified the nearby genes whose RNA expression was altered in AD: ANK1, CDH23, DIP2A, RHBDF2, RPL13, SERPINF1 and SERPINF2. Our analyses suggest that these DNA methylation changes may have a role in the onset of AD given that we observed them in presymptomatic subjects and that six of the validated genes connect to a known AD susceptibility gene network.

Methylomic profiling implicates cortical deregulation of ANK1 in Alzheimer's disease.

Alzheimer's disease (AD) is a chronic neurodegenerative disorder that is characterized by progressive neuropathology and cognitive decline. We performed a cross-tissue analysis of methylomic variation in AD using samples from four independent human post-mortem brain cohorts. We identified a differentially methylated region in the ankyrin 1 (ANK1) gene that was associated with neuropathology in the entorhinal cortex, a primary site of AD manifestation. This region was confirmed as being substantially hypermethylated in two other cortical regions (superior temporal gyrus and prefrontal cortex), but not in the cerebellum, a region largely protected from neurodegeneration in AD, or whole blood obtained pre-mortem from the same individuals. Neuropathology-associated ANK1 hypermethylation was subsequently confirmed in cortical samples from three independent brain cohorts. This study represents, to the best of our knowledge, the first epigenome-wide association study of AD employing a sequential replication design across multiple tissues and highlights the power of this approach for identifying methylomic variation associated with complex disease.

Polarization of the effects of autoimmune and neurodegenerative risk alleles in leukocytes.

To extend our understanding of the genetic basis of human immune function and dysfunction, we performed an expression quantitative trait locus (eQTL) study of purified CD4(+) T cells and monocytes, representing adaptive and innate immunity, in a multi-ethnic cohort of 461 healthy individuals. Context-specific cis- and trans-eQTLs were identified, and cross-population mapping allowed, in some cases, putative functional assignment of candidate causal regulatory variants for disease-associated loci. We note an over-representation of T cell-specific eQTLs among susceptibility alleles for autoimmune diseases and of monocyte-specific eQTLs among Alzheimer's and Parkinson's disease variants. This polarization implicates specific immune cell types in these diseases and points to the need to identify the cell-autonomous effects of disease susceptibility variants.

5'RNA-Seq identifies Fhl1 as a genetic modifier in cardiomyopathy.

The transcriptome is subject to multiple changes during pathogenesis, including the use of alternate 5' start-sites that can affect transcription levels and output. Current RNA sequencing techniques can assess mRNA levels, but do not robustly detect changes in 5' start-site use. Here, we developed a transcriptome sequencing strategy that detects genome-wide changes in start-site usage (5'RNA-Seq) and applied this methodology to identify regulatory events that occur in hypertrophic cardiomyopathy (HCM). Compared with transcripts from WT mice, 92 genes had altered start-site usage in a mouse model of HCM, including four-and-a-half LIM domains protein 1 (Fhl1). HCM-induced altered transcriptional regulation of Fhl1 resulted in robust myocyte expression of a distinct protein isoform, a response that was conserved in humans with genetic or acquired cardiomyopathies. Genetic ablation of Fhl1 in HCM mice was deleterious, which suggests that Fhl1 transcriptional changes provide salutary effects on stressed myocytes in this disease. Because Fhl1 is a chromosome X-encoded gene, stress-induced changes in its transcription may contribute to gender differences in the clinical severity of HCM. Our findings indicate that 5'RNA-Seq has the potential to identify genome-wide changes in 5' start-site usage that are associated with pathogenic phenotypes.